Golden Gate Assembly Protocol - Revision history

Eckdahl at 16:15, 9 February 2017

2017-02-09T16:15:41Z

Eckdahl at 16:12, 9 February 2017

2017-02-09T16:12:00Z

Eckdahl at 15:39, 9 February 2017

2017-02-09T15:39:25Z

Eckdahl at 19:44, 1 March 2016

2016-03-01T19:44:04Z

Eckdahl at 19:22, 1 March 2016

2016-03-01T19:22:26Z

Eckdahl at 19:08, 1 March 2016

2016-03-01T19:08:28Z

Eckdahl at 19:06, 1 March 2016

2016-03-01T19:06:53Z

Eckdahl: Created page with "Golden Gate Assembly Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL promoter or other insert :1 µL 10X Promega Ligase Buffer :6 µL dH₂O :0..."

2015-06-01T19:27:45Z

Created page with "Golden Gate Assembly Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL promoter or other insert :1 µL 10X Promega Ligase Buffer :6 µL dH<sub>2</sub>O :0..."

New page

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL promoter or other insert

:1 µL 10X Promega Ligase Buffer

:6 µL dH<sub>2</sub>O

:0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL total volume

Turn on the PCR machine. Put tube into machine. Program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

@@ Line 8: / Line 8: @@
 :1 µL 10X Promega Ligase Buffer
-:6 µL dH<sub>2</sub>O
+:6.9 µL dH<sub>2</sub>O
 :0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)
-: 0.5 µL T4 DNA Ligase to the mixture
+:0.5 µL T4 DNA Ligase to the mixture
-:10 µL total volume
+:10.9 µL total volume

@@ Line 19: / Line 19: @@
 Turn on the thermal cycler machine. Put tube into machine. For BsaI or BbsI GGA, program it for the following cycles:
-:• 20 cycles
+:• 30 cycles
 :• 37°C for 1 minute
@@ Line 25: / Line 25: @@
 :• 16°C for 1 minute
-:• 22°C holding temperature
+:• 37°C for 15 minutes (after 30 cycles)

@@ Line 2: / Line 2: @@
 GGA mixture contains:
-:1 µL (50 ng) Plasmid
+:1 µL (20 ng) Plasmid
 :1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)

@@ Line 38: / Line 38: @@
 :• 16°C for 1 minute
-:• 22°C holding temperature
+:• 10°C holding temperature
 After GGA, the DNA is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

← Older revision		Revision as of 19:08, 1 March 2016
Line 17:		Line 17:


−	Turn on the ~~PCR~~ machine. Put tube into machine. Program it for the following cycles:	+	Turn on the thermal cycler machine. Put tube into machine. Program it for the following cycles:

	:• 20 cycles		:• 20 cycles

@@ Line 17: / Line 17: @@
-Turn on the thermal cycler machine. Put tube into machine. Program it for the following cycles:
+Turn on the thermal cycler machine. Put tube into machine. For BsaI or BbsI GGA, program it for the following cycles:
 :• 20 cycles
@@ Line 29: / Line 40: @@
 :• 22°C holding temperature
-This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
+After GGA, the DNA is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
 Transformation of GGA
 For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.